Process for isolating hepatitis B antigen

ABSTRACT

An improved process for the preparation of hepatitis B antigen by subjecting plasma of hepatitis B donors to isopycanic banding wherein the step gradient is NaBr. Faster and more economical processing is obtained by multiple loading of the step gradient, either NaBr or conventional gradients.

BACKGROUND OF THE INVENTION

This invention relates to hepatitis B and, more particularly, to avaccine for hepatitis B and to a method for purifying hepatitis Bantigen for use as a vaccine.

Hepatitis B is one of the types of viral hepatitis which results in asystemic infection with the principal pathologic changes occurring inthe liver. This disease affects mainly adults and is maintained chieflyby transfer of infection from long term carriers of the virus. Usualmethods of spread are by blood transfusion, contaminated needles andsyringes, through skin breached by cuts or scratches, by unsterilizeddental instruments as well as by saliva, venereal contact or exposure toaerosolized infected blood.

The incubation period of type B hepatitis is relatively long: from 6weeks to 6 months may elapse between infection and the onset of clinicalsymptoms. The illness usually begins with fatigue and anorexia,sometimes accompanied by myalgia and abdominal discomfort. Laterjaundice, dark urine, light stools and tender hepatomegaly may appear.In some cases, the onset may be rapid, with appearance of jaundice earlyin association with fever, chills and leukocytosis. In other casesjaundice may never be recognized and the patient may be aware only of a"flu-like" illness. It is estimated that the majority of hepatitisinfections result in a mild, anicteric illness.

It is, accordingly, an object of the present invention to provide animproved method for extracting hepatitis B surface antigen. Anotherobject is to provide a faster and more economical method of extractingantigen. A further object is to provide antigen which is free of addedcesium or potassium. These and other objects of the present inventionwill be apparent from the following description.

SUMMARY OF THE INVENTION

Plasma of hepatitis B donors is subjected to an isopycnic bandingwherein the gradient is NaBr. Faster and more economical processing ofthe plasma is obtained by multiple loading of the step gradient whetherthe step gradient be NaBr or a conventional gradient.

DETAILED DESCRIPTION

The starting material for the purified hepatitis B surface antigen (HB₂Ag) of the present invention is plasma obtained from hepatitis B donors,e.g., by plasmaphoresis. The level of antigen may be measured in knownmanner by radioimmune assay, passive hemagglutination or complementfixation. The plasma is cooled and the cryoprecipitate which forms isremoved by light centriguation. The HB_(s) Ag in the resulting clarifiedplasma is isolated by an isopycnic banding step followed by a rate zonalbanding step.

In isopycnic banding the partially purified concentrate is contactedwith a liquid medium having a density gradient therein which includesthe density of the specific antigen being isolated. The liquid medium isthen subjected to ultracentrifugation to attain an equilibriumdistribution of the serum components through the density gradientaccording to their individual densities. Successive fractions of themedium are displaced and those containing the desired antigen, i.e. thefractions having a density of from about 1.21 to about 1.24 g/cc, areseparated. The application of this technique to the purification ofHB_(s) Ag is described in German Specification No. 2,049,515 and U.S.Pat. No. 3,636,191. The concentrations of the solutions forming thegradient are selected so as to encompass the density range of from about1.0 to about 1.41 g/cc. The liquid medium may be employed in the form ofa linear gradient or a step gradient. Preferably it is employed in theform of a step gradient due to its inherent higher capacity forfractionation.

In rate zonal banding the partially purified concentrate is subjected toultracentrifugation in contact with a liquid medium having a densitygradient therein, but this time using the rate zonal technique, i.e., ata rate and for a period such that equilibrium is not attained, theHB_(s) Ag and other residual serum components being distributed throughthe medium according to their sedimentation coefficients in the medium.The concentrations of the solutions forming the step gradient areselected so as to encompass the density range of from about 1.0 to about1.28 g/cc. The rate zonal step is carried out until the HB_(s) Agresides in the 1.13 to 1.16 density region. At this point the HB_(s) Agis separated from the bulk of the crude plasma proteins and, mostsignificantly, is also separated from the macroglobulin complement ofthe plasma. If the rate zonal step is carried out such that the desiredHB_(s) Ag antigen reaches its equilibrium position, i.e., about 1.18 toabout 1.20 g/cc, it has been found that a plasma macroglobulin fractionwill appear as a contaminant in the desired HB_(s) Ag antigen fraction.

The liquid media used in the isopycnic banding and rate zonal steps maybe any density gradient in the appropriate ranges. Prior art solutes forsuch solutions include. e.g. sucrose, potassium bromide, cesiumchloride, potassium tartrate and the like.

The isopycnic banding step is conveniently carried out in a centrifuge,for example, Electronucleonics-K, by filling the stationary rotor withsaline solution, then successively displacing the saline solutionupwards with aliquots of a liquid medium solution of increasing densityuntil a step gradient is formed. The plasma is introduced at the top ofthe rotor displacing some of the highest density solution from thebottom. Typically, the volume of plasma is from about 15% to about 40%that of the step gradient. The centrifuge is brought up to speed througha programmed speed control system which prevents mixing during theinitial reorientation phase. When equilibrium is attained and theproduct is in its proper density position, the rotor is slowed downthrough the same system to prevent mixing upon reorientation to theoriginal configuration. Then the gradient is drained from below and theproper density cut collected. A similar technique is used in the ratezonal banding. The proper density cut from the rate zonal banding is thedesired concentrate of hepatitis B antigen.

Due to the samll size, approximately 20 nm, of HB_(s) Ag the isopycnicbanding step is quite time consuming, requiring about 18 hours ofcentrifuging. As a result, even operating 24 hours a day, 7 days a week,it is possible to prepare only about 4 batches of clarified plasma percentrifuge. Productivity can be increased, of course, by utilizingadditional centrifuges. This involves a tremendous capital investment,however, as each centrifuge costs about $100,000.

It has now been found that substantial increases in productivity andsubstantially reduced operating costs are obtained by multiple loadingof the isopycnic banding gradient. Multiple loading means subjecting asample of clarified plasma containing HB_(s) Ag to isopycnic bandingconditions for a time sufficient to permit substantially all of theHB_(s) Ag in the clarified plasma to pass into the gradient butinsufficient to achieve equilibrium, and repeating this step at leastonce with an additional sample of clarified plasma containing HB_(s) Ag,before continuing the isopycnic banding conditions for a time sufficientto achieve equilibrium. If desired, a gradient may be loaded with up toabout 6 samples of clarified plasma. As the time required for the HB_(s)Ag in the clarified plasma to enter the gradient is only a fraction ofthat required to reach equilibrium, and as the subsequent time requiredto reach equilibrium is the same whether the gradient is single ormultiply loaded, substantial savings in time and reductions in unitprocessing costs are obtained.

While the increased productivity and reduced costs of the multiplebanding technique of the present invention may be achieved with anysuitable gradient, preferably the gradient is sodium bromide.

The isopycnic is carried out to equilibrium by centrifuging at fromabout 40,000 × g to about 80,000 × g for about 10 hours or beyond. Ithas been found, however, that by centrifuging the plasma for about 4hours substantially all of the HB_(s) Ag is caused to move into theisopycnic banding gradient. Then the sample of spent plasma is removedand a fresh sample of plasma equal in volume to the first sample islayered onto the gradient. Centrifuging may then be continued aspreviously for about 10 hours or beyond to cause the HB_(s) Ag in bothsamples to move into the equilibrium density region of the gradient(1.21 to about 1.24 g/cc) to complete the banding. Alternatively thecentrifuging may be continued for 4 hours, the spent plasma removed anda third sample of fresh plasma layered onto the gradient. This multipleloading procedure may be repeated six or even more times beforecompleting the banding by centrifuging for about 18 hours.

The ratio of the charge (plasma) volume to the gradient volume is fromabout 1:3 to about 1:6. When a single plasma charge is applied to thegradient and centrifuged under isopycnic banding conditions (e.g. forfrom about 16 to about 20 hours at 30,000 rpms in the K-II centrifuge)the resulting product generally will have a protein content ofapproxmately 4-10 mg/ml in a volume of 1.0 liter, depending on theamount of protein in the original plasma.

When a double charge of plasma is applied to the gradient andcentrifuged under isopycnic banding conditions, (for from about 16 toabout 20 hours at 30,000 rpms) the resulting product will have a proteincontent which is additive for the charges employed, typically from about8-20 mg/ml in a volume of 1.0 liter, depending on the amount of proteinin the original plasma. The level of protein increases in this mannerfor each subsequent charge of plasma applied to the gradient.

The product is then subjected to a rate zonal banding. The rate zonalbanding is carried out until the HB_(s) Ag is in the density range offrom about 1.13 to about 1.16 g/cc. Typically this takes for from about16 hours to about 20 hours, preferably for from about 17 to about 18hours, at from about 30,000 × g to about 60,000 × g.

According to one aspect of the present invention the gradient is formedof sodium bromide whether or not the multiple loading technique is used.In contrast to heretofore used materials sodium bromide has definiteadvantages. The solubility of sodium bromide allows the use of highdensity solutions in the formation of gradients at refrigeratortemperatures (2°-6° C). There are definite economic advantages to usingsodium bromide over a salt such as cesium chloride as well as not havingto contend with the problem of human toxicity from residual and HB_(s)Ag bound cesium ions. In sodium bromide gradient any ions bound to theHb_(s) Ag, due to biophysical characteristics, will be a sodium saltwhich is very compatible with the human bioligical system and does notpresent a toxicity problem.

The biophysical characteristics of the HB_(s) Ag are well documented [J.Clinical Investigation 52, 1176 (1973), J. of Virology 10, 469 (1972)]as a negatively charged particle. In the presence of a very highconcentration of positively charged sodium ions there is formed asodium-HB_(s) Ag salt molecule. This type of molecule is compatible withthe human biological system. In contrast to prior art products, theHB_(s) Ag of the preferred modes carrying out the present invention issubstantially free of other cations, particularly added cesium andpotassium ions.

The superior solubility of NaBr at lowered temperatures with respect toKBr permits the use of lowered temperatures more conductive to stabilityof biological materials. The use of a step gradient rather than a lineargradient is preferred as it accumulates impurities at the stepboundaries and permits processing a larger volume of plasma in a singlegradient.

The antigen of the present invention is useful per se as an antigen forhepatitis B and can be used as described in U.S. Pat. No. 3,636,191. TheHB_(s) Ag antigen of the present invention is a highly purified product.The isopycnic banding step results in about a 100 fold purificationHB_(s) Ag relative to normal plasma protein. The rate zonal step resultsin about a further 20 fold purification of HB_(s) Ag relative to normalplasma protein. The combination of the two steps result in about a 2000fold purification of HB_(s) Ag relative to normal plasma protein. Theresulting product has been shown to be substantially free of blood groupsubstances A and B as measured by serological and electrophoresistechniques. In addition, the antigen of the present invention can beused as the starting material for the hepatitis B antigen of copendingapplication Ser. No. 577,483, filed 14 May 1975.

The following examples illustrate the present invention without,however, limiting the same thereto.

EXAMPLE 1

The rotor of a centrifuge, Electronucleonics K, is filled with 8,400 mlof phosphate buffer. After running the rotor up to 10,000 rpm to degasthe system, the following step gradient is pumped into the bottom of thestationary rotor:

1. 2,400 ml of 10% NaBr, ρ=1.08

2. 1,000 ml of 20% NaBr, ρ=1.17

3. 1,500 ml of 30% NaBr, ρ=1.28

4. 3,500 ml of 40% NaBr, ρ=1.41

Plasma containing Australia antigen (HB_(s) Ag), 1,750 ml, is pumpedinto the top of the stationary rotor displacing 1,750 ml of 40% NaBrfrom the bottom of the rotor. The rotor is accelerated to 30,000 rmp andrun at this speed for 18 hours. After stopping the rotor 500 ml ofHB_(s) Ag rich material in the 1.21-1.24 density region, is collectedand dialyzed against phosphate buffer.

The rotor is then filled with phosphate buffer, degassed as above, andthe following step gradient pumped into the bottom of the stationaryrotor:

1. 2,400 ml of 5% sucrose, ρ=1.02

2. 1,750 ml of 15% sucrose, ρ=1.06

3. 1,750 ml of 25% sucrose, ρ=1.10

4. 2,500 ml of 50% sucrose, ρ=1.23

The HB_(s) Ag rich material from the NaBr isopycnic banding step, 500ml, is pumped into the rotor top displacing 500 ml. of 50% sucrose outthe rotor bottom. The rotor is then run at 28,000 rpm for 18 hours.After stopping the rotor, 500 ml of HB_(s) Ag rich material in the1.135-1.165 density region is collected.

EXAMPLE 2

The rotor of a centrifuge, Electronucleonics K, is filled with 8,400 mlof phosphate buffer. After running the rotor up to 10,000 rpm to degasthe system, the following step gradient is pumped into the bottom of thestationary rotor:

1. 2,400 ml of 10% NaBr, ρ=1.08

2. 1,000 ml of 20% NaBr, ρ=1.17

3. 1,500 ml of 30% NaBr, ρ=1.28

4. 3,500 ml of 40% NaBr, ρ=1.41

Plasma containing HB_(s) Ag, 1,750 ml, is pumped into the top of thestationary rotor displacing 1,750 ml of 40% NaBr from the bottom of therotor. The rotor is accelerated to 30,000 rpm and run at this speed for4 hours. The rotor is then stopped and 1,750 of 40% NaBr are pumped intothe bottom of the rotor forcing the plasma out the top. An additional1,750 ml of fresh plasma containing HB_(s) Ag are pumped into the top ofthe rotor displacing an equal volume of 40% NaBr out the bottom of therotor. The rotor is then run at 30,000 rpm for 18 hours. After stoppingthe rotor 1,000 ml of HB_(s) Ag rich material in the 1.21-1.24 densityregion is collected and dialyzed against phosphate buffer.

The rotor is then filled with phosphate buffer, degassed as above, andthe following step gradient pumped into the bottom of the stationaryrotor:

1. 2,400 ml of 5% sucrose, ρ=1.02

2. 1,750 ml of 15% sucrose, ρ=1.06

3. 1,750 ml of 25% sucrose, ρ=1.10

4. 2,500 ml of 50% sucrose, ρ=1.23

The HB_(s) Ag rich material from the NaBr isopycnic banding step, 1,000ml, is pumped into the rotor top displacing 1,000 ml. of 50% sucrose outthe rotor bottom. The rotor is then run at 28,000 rpm for 18 hours.After stopping the rotor, 1,000 ml of HB_(s) Ag rich material in the1.135-1.165 density region is collected.

EXAMPLE 3

The rotor of a centrifuge, Electronucleonics K, is filled with 8,400 mlof phosphate buffer. After running the rotor up to 10,000 rpm to degasthe system, the following step gradient is pumped into the bottom of thestationary rotor:

1. 2,400 ml of 10% NaBr, ρ=1.08

2. 1,000 ml of 205 NaBr, ρ=1.17

3. 1,500 ml of 30% NaBr, ρ=1.28

4. 3,500 ml of 40% NaBr, ρ=1.41

Plasma containing HB_(s) Ag, 1,750 ml, is pumped into the top of thestationary rotor displacing 1,750 ml of 40% NaBr from the bottom of therotor. The rotor is accelerated to 30,000 rpm and run at this speed for4 hours. The rotor is then stopped and 1,750 ml of 40% NaBr are pumpedinto the bottom of the rotor forcing the plasma out the top. Anadditional 1,750 ml of fresh plasma containing HB_(s) Ag are pumped intothe top of the rotor displacing an equal volume of 40% NaBr out thebottom of the rotor. The rotor is accelerated to 30,000 rpm and run atthis speed for 4 hours. The rotor is then stopped and a third charge of1,750 ml of fresh plasma containing HB_(s) Ag are pumped into the top ofthe rotor displacing an equal volume of 40% NaBr out the bottom of therotor. The rotor is then run at 30,000 rpm for 18 hours. After stoppingthe rotor, 1,500 ml of HB_(s) Ag rich material in the 1.21-1.24 densityregion is collected and dialyzed against phosphate buffer.

The rotor is then filled with phosphate buffer, degassed as above, andthe following step gradient pumped into the bottom of the stationaryrotor:

1. 2,400 ml of 5% sucrose, ρ=1.02

2. 1,750 ml of 15% sucrose, ρ=1.06

3. 1,750 ml of 25% sucrose, ρ=1.10

4. 2,500 ml of 50% sucrose, ρ=1.23

The HB_(s) Ag rich material from the NaBr isopycnic banding step, 1,500ml, is pumped into the rotor top displacing 1,500 ml of 50% sucrose outthe rotor bottom. The rotor is then run at 28,000 rpm for 18 hours.After stopping the rotor 1,500 ml of HB_(s) Ag rich material in the1.135 1.165 density region is collected.

EXAMPLE 4

The following table shows the marked increase in yield per unit of timewhen using the multiple loading technique of the present invention(Examples 2 and 3) as compared with single loading (Example 1).

    ______________________________________                                                       Total isopycnic                                                                           % Increase                                                                             % Increase                                      Yield    and rate zonal                                                                            in time (with                                                                          in yield (with                            Exam- (ml) of  centrifuging                                                                              respect to                                                                             respect to                                ple   HB.sub.s Ag                                                                            time (hours)                                                                              Example 1)                                                                             Example 1)                                ______________________________________                                        1      500        36       --       --                                        2     1,000       40       11.1%    100%                                      3     1,500       44       22.2%    200%                                      ______________________________________                                    

What is claimed is:
 1. In a process for concentrating HB_(s) Ag fromclarified plasma of human hepatitis B donors, the improvement comprisingsubjecting the clarified plasma to isopycnic banding in sodium bromidedensity gradient and recovering a fraction rich in HB_(s) Ag.
 2. Aprocess according to claim 1 wherein the density gradient is a stepgradient.
 3. In a process for purifying HB_(s) Ag from a plasma that hasbeen subjected to an isopycnic banding in sodium bromide gradient, theimprovement comprising subjecting the isopycnic banding fraction havinga density region of from about 1.21 g/cc to about 1.24 g/cc to a ratezonal fractionation until the HB_(s) Ag is found in the density regionof from about 1.13 g/cc to about 1.16 g/cc.
 4. In a process according toclaim 1 the improvement comprising subjecting the isopycnic bandingproduct containing HB_(s) Ag to a rate zonal fractionation until theHB_(s) Ag is found in the density region of from about 1.13 g/cc toabout 1.16 g/cc.